PureYield™ Plasmid Midiprep System From Promega

Many protein expression procedures require large amounts of pure plasmid DNA. Over the years, a number of kits have been developed to streamline the isolation of plasmid DNA from large bacterial cultures, and the speed and simplicity of the kits are improving as time goes on. The PureYield™ Plasmid Midiprep System from Promega has been designed specifically for isolating DNA for in vitro transcription and translation and cell culture expression of proteins. Features of the kit include a new form of silica membrane for DNA isolation, an endotoxin removal step and a faster isolation time of 30 minutes (depending on the protocol used). Up to 200 µg of DNA can be harvested from 50 ml of culture, and membrane binding and wash steps can be done using a vacuum protocol, centrifugation or a combination of both. Kits are available to isolate DNA from 4, 25 and 100 x 50 ml cultures.

Each kit contains Cell Resuspension Solution, Cell Lysis Solution, Neutralization Solution and Column Wash. Also included are Endotoxin removal Wash, Nuclease-Free Water for elution and a set of PureYield™ Clearing and Binding Columns. The protocol is simple and similar to other Promega kits for DNA isolation. An appropriate amount of overnight bacterial culture is pelleted, and vortexed in Resuspension Solution. Cells are lysed, and the lysate neutralized with 3 minute incubations times between each step. Next, the lysate is cleared using the Clearing Column, and the DNA on the Binding column for washing. This can be achieved in a number of steps by centrifugation using a centrifuge with a swinging bucket rotor as the columns are designed to fit inside a standard 50 ml plastic tube. Alternately, the two columns can be stacked and the lysate pulled through in series using a vacuum manifold (not supplied). A third method is a combination of lysate clearing by centrifugation and washing using vacuum. Each protocol has benefits – the vacuum protocol is fastest, however DNA yields may be higher from the centrifugation protocol due to more efficient lysate clearing. A combination protocol is provided which is designed to get the best of both methods. After the Endotoxin Removal Wash and a normal Column Wash, the DNA is eluted in 600 µl Nuclease-Free water by centrifugation.

We have used a number of large-volume DNA isolation kits on plasmids from 4 to 13 kb in our lab, and the PureYield™ Plasmid Midiprep System is so far the fastest and simplest way of isolating high-copy number plasmids from culture. One drawback of the system is to achieve decent yields from low-copy number plasmids you need larger volume cultures. If working with cultures from 50-250 ml the manufacturer recommends using higher volumes of Cell Resuspension, Lysis and Neutralization Solution to ensure proper processing of the culture. Of course, this means the solutions will run out before the columns do, requiring more to be purchased or made up according to the instructions supplied by Promega. We primarily use plasmid DNA for transfection in mammalian and insect cells, and find Promega kits consistently provide good quality DNA, free of endotoxins. While the plasmid DNA can also be used straight away for in vitro transcription, we have found better yields when the DNA is phenol-chloroform extracted first.